基因工程菌培养过程中乙酸的积累及其对工程菌生长和产物表达的影响

在rIL-2工程菌K802(pLY-4)高密度培养中发现培养液中有大量代谢副产物乙酸积累,乙酸的存在对工程菌的生长和产物的表达均有明显的抑制作用,这种抑制作用是制约工程菌高密度培养的重要因素,为了减小这种抑制作用,研究了培养基pH与乙酸抑制作用的关系,发现适当提高培养基pH值,能有效地减小乙酸的抑制作用;高密度培养时,提高培养基的pH后,虽然仍有大量乙酸积累,但产物的表达水平和菌密度都有一定的提高     

乙酸对重组大肠杆菌生长及个源基因表达的影响

在重组基因工程菌ＤＨ５α（ＰＧ－ＦＧＦ）的高密度培养过程中,发现培养液中有大量代谢副产物－乙酸的产生和积累,乙酸的存在抑制了工程菌的生长及外源的表达。研究民乙酸在Ｍ９培养基中对工程菌ＤＨ５α（ＰＧ－ＦＧＦ）及生长外源基因表达的影响。结果表明,乙酸的存在不仅导致重组菌生长速率的降低及延迟期的增长,而且对外源基因产物的表达具有强烈的抑制作用,这为该工程菌的高密度培养及外源基因产物的高表达打下了基础。     

乙酸对重组大肠杆菌生长及外源基因表达的影响

在重组基因工程菌ＤＨ５α（ＰＧ－ＦＧＦ）的高密度培养过程中,发现培养液中有大量代谢副产物－乙酸的产生和积累,乙酸的存在抑制了工程菌的生长及外源基因的表达。研究了乙酸在Ｍｇ培养基中对工程菌ＤＨ５α（ＰＧ－ＦＧＦ）生长及外源基因表达的影响。结果表明,乙酸的存在不仅导致重组菌生长速率的降低及延迟期的增长,而且对外源基因产物的表达具有强烈的抑制作用,这为该工程菌的高密度培养及外源基因产物的高表达打下了基础。     

比生长速率与培养基成份对rIL-2工程菌生长和表达的影响

基因工程菌高密度培养对提高单位体积培养物的产量,提高生产能力,减轻下游负担均有积极意义。本文对由PL-λCI_(857)温度诱导控制系统控制的rIL 2工程菌的高密度培养,及在高密度时实现较高表达的方案进行了初步研究。发现该菌的高密度生长时有乙酸积累,在升温诱导后乙酸积累加快,乙酸的存在对菌体生长和产物表达均有明显的抑制作用,这种抑制     

工程菌发酵过程中乙酸的形成及其控制方法

工程菌高密度培养是获得外源基因表达产物的重要手段,但高密度培养的主要障碍之一是代谢副产物乙酸的积累。随着发酵培养密度的提高,乙酸的积累也增加,并直接影响菌体的生长和外源蛋白的表达,逐渐成为制约工程菌高密度培养的重要因素。Jensen等报道当培养液中乙酸浓度大于6g/L时,乙酸会明显抑制菌体生长;当乙酸浓度大于2.4g/L时,会显著降低比产率。Konstan等报道培养液中乙酸浓度大于15g/L时菌体生长就完全停止了。Boon等人利用从     

大肠杆菌乙酸代谢突变株的选育和特性研究

在大肠杆菌高密度培养中 ,因代谢副产物乙酸积累 ,导致抑制菌体的生长和产物表达的下降。为减小乙酸的抑制作用 ,采用60 Co诱变处理大肠杆菌JM1 0 1 ,结合连续培养 (含乙酸钠选择压力 )定向富集方法 ,选育到一株乙酸耐受性增强的菌株JL3。该菌株表现出明显的乙酸耐受性的提高 ,在含有 1 0 g/L乙酸钠的MA培养基中 ,菌体生长和葡萄糖消耗速率都有较大程度提高 ,并且具有良好的遗传稳定性     

rIL-2工程菌诱导阶段细胞再循环培养的研究

为了减少rIL-2工程菌高密度培养时乙酸的积累,在诱导阶段对该工程菌进行细胞再循环培养的研究,比较了细胞再循环补料液、pH、细胞循环培养时间段对工程菌的生长及rIL-2表达的影响。结果表明在菌密度D_(600)为50时,细胞再循环补料液中酵母抽提物与胰蛋白胨浓度为发酵培养基的5倍就能满足rIL-2表达的需求,同时选择诱导后4～6h之间的细胞再循环培养能有效地防止乙酸的过高积累并减少营养物质的损失,有利于rIL-2的表达。根据以上研究结果得到了rIL-2工程菌诱导阶段细胞再循环培养方法,使得在诱导前菌密度D_(600)为50左右时rIL-2的表达水平约为40％。     

RP-HPLC法测定工程菌发酵液中的乙酸含量

随着基因工程的发展,工程菌发酵工艺越来越受到重视,工程菌在含有葡萄糖的培养基发酵过程中,往往有乙酸产生。乙酸的产量与比生长速率有关,乙酸的存在对工程菌的生长及表达物有影响。本文采用了RP-HPLC法,选用简便的样品预处理方法,测定了温度诱导型IL-2工程菌发酵液中的乙酸量,得到了较好的分离效果。发现该菌在培养过程中确有乙酸积累,并在升温诱导后乙酸量急剧增加。 1 材料和方法 1.1 试剂     

重组大肠杆菌高密度培养研究进展

大肠杆菌是基因工程中常用的宿主菌,许多有价值的多肽和蛋白在大肠杆菌中已成功地进行了表达,表达水平有些高达细胞总蛋白的３０％以上,为了提高单位体积设备产物的产量,除了提高表达水平外,还需提高重组大肠杆菌的培养密度。大肠杆菌高密度培养的早期工作主要是以宿主菌为模型进行研究的,而近年来,重组大肠杆菌高密度高表达的研究已经有了较大进展。本文着重综述了培养基成分、溶氧、比生长速率和代谢副产物乙酸等因素对工程菌生长和表达的影响,及提高菌密度和表达水平的培养方法。     

霍乱毒素B亚单位工程菌MM2的表达与lac启动子的关系

研究了不同碳源(葡萄糖、乳酸和乙酸)以及IPTG诱导对工程菌MM2表达霍乱毒素B亚单位(CTB)的影响,ctb基因位于lac启动子的下游。在YC培养基中分别加入0.048mol/L的葡萄糖、0.102mol/L的乳酸和0.167mol/L的乙酸,它们在完全氧化后可产生相同的能量。结果表明,加入葡萄糖会大幅度降低ctb基因的表达水平,其原因和培养过程中pH值下降有关;加入乳酸可提高ctb基因表达水平1.15倍,且不抑制菌体生长;加入乙酸可提高ctb基因的表达水平0.9倍,但对菌体生长有抑制作用。不同时间及不同浓度的IPTG诱导未能提高ctb基因的表达水平,说明lac启动子对ctb基因的表达没有影响或影响很小。     

Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor

High-cell-density cultivations of Escherichia coli K12 in a dialysis reactor with controlled levels of dissolved oxygen were carried out with different carbon sources: glucose and glycerol. Extremely high cell concentrations of 190 g/l and 180 g/l dry cell weight were obtained in glucose medium and in glycerol medium respectively. Different behaviour was observed in the formation of acetic acid in these cultivations. In glucose medium, acetic acid was formed during the earlier phase of cultivation. However, in glycerol medium, acetic acid formation started later and was particularly rapid at the end of the cultivation. In order to estimate the influence of acetic acid during these high-cell-density cultivations, the inhibitory effect of acetic acid on cell growth was investigated under different culture conditions. It was found that the inhibition of cell growth by acetic acid in the fermentor was much less than that in a shaker culture. On the basis of the results obtained in these investigations of the inhibitory effect of acetic acid, and the mathematical predictions of cell growth in a dialysis reactor, the influence of acetic acid on high-cell-density cultivation is discussed. Received: 20 May 1997 / Received revision: 12 August 1997 / Accepted: 25 August 1997     

High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii

The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring.     

Isolation of a Strain of Clostridium thermoaceticum Capable of Growth and Acetic Acid Production at pH 4.5

Using a series of pH controlled batch fermentations operated in a fed-batch mode and adaptation and selection techniques where pH and acetic acid provided the selective pressures, we isolated a culture of Clostridium thermoaceticum that can grow and produce acetic acid at pH 4.5. At pH 4.5 the fastest mass doubling time was 36 h, and the highest acetic acid concentration reached was 4.5 g/liter. Generally, as the pH was decreased from 6.0 and the initial acetic acid concentration increased, the mass doubling time increased, and the final acetic acid concentration decreased. These observations can be explained in terms of inhibition by the free acetic acid concentration at a given pH, relative to the total acetic acid concentration (free acid plus acetate ion). We have thus reached one of the criteria determined by us to be required for an economically viable fermentation acetic acid process, i.e., pH 4.5. A second requirement for a mass doubling time of about 7 h (0.1/h dilution rate) can probably be reached by selection in continuous culture. The final requirement for an acetic acid concentration of 50 g/liter will be the most difficult to achieve in view of the organism's sensitivity to low concentrations of free acetic acid.     

可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

On-line extraction of volatile fatty acids in acidogenic chemostat culture using a supported liquid membrane

An on-line extraction of volatile fatty acids (acetic and butyric acids) from acidogenic fermentation in chemostat cultures using a supported liquid membrane (SLM) was investigated in order to overcome end-product inhibition. By using SLM, the high-cell-density retaining dilution rate of the chemostat could be increased, thus enhancing the microbial acidogenesis. To further understand this phenomenon, the growth and extraction kinetics were studied. The effect of substrate concentration was found to obey the Haldane equation. Regarding the inhibition by volatile fatty acids, it turned out that undissociated butyric acid rather than acetic acid severely inhibited the growth, corresponding to non-competitive kinetics. The extraction rates of the acids were proportional to their undissociated concentration as well as to the SLM area/broth volume, and butyric acid extraction was easier than that of acetic acid.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Sulfidogenesis in low pH (3.8-4.2) media by a mixed population of acidophilic bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8-4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8-4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.